Materials Science and Engineering C 46 (2015) 463–469
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Green synthesis and characterization of silver nanoparticles using alcoholic flower extract of Nyctanthes arbortristis and in vitro investigation of their antibacterial and cytotoxic activities Nayanmoni Gogoi a,c, Punuri Jayasekhar Babu a,b, Chandan Mahanta a,d, Utpal Bora a,b,⁎ a
Biotech Hub, Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India Biomaterials and Tissue Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India c Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India d Department of Civil Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India b
a r t i c l e
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Article history: Received 30 June 2014 Received in revised form 19 September 2014 Accepted 23 October 2014 Available online 24 October 2014 Keywords: Nyctanthes arbortristis Spectroscopy Crystal growth of silver nanoparticles FTIR Electron microscopy Nanocrystalline materials Antibacterial Cell viability
a b s t r a c t Here we report the synthesis of silver nanoparticles using ethanolic flower extract of Nyctanthes arbortristis, UVvisible spectra and TEM indicated the successful formation of silver nanoparticles. Crystalline nature of the silver nanoparticles was confirmed by X-ray diffraction. Fourier Transform Infra-Red Spectroscopy analysis established the capping of the synthesized silver nanoparticles with phytochemicals naturally occurring in the ethanolic flower extract of N. arbortristis. The synthesized silver nanoparticles showed antibacterial activity against the pathogenic strain of Escherichia coli MTCC 443. Furthermore, cytotoxicity of the silver nanoparticles was tested on mouse fibroblastic cell line (L929) and found to be non-toxic, which thus proved their biocompatibility. Antibacterial activity and cytotoxicity assay carried out in this study open up an important perspective of the synthesized silver nanoparticles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silver nanoparticles (AgNPs) have been the focus of increasing research interest in the past decade. In the recent times, AgNPs serve as an excellent candidate for most of the therapeutic purposes [1]. Studies on AgNPs have confirmed their potential application in many fields of science [2]. AgNPs have shown positive results in wound healing, retinal therapies, DNA sequencing and pharmaceuticals along with other conventional uses like in electronics, optics, catalysis and Raman scattering [3–11]. The fundamental uses of AgNPs have been highly recognized in water treatment process. Contemporary AgNP filters are effective and utilized against contamination in water treatment and filtration processes [12,13]. The efficiency of such filters is strongly attributed to the antimicrobial properties acquired by AgNPs [14]. Green synthesis of metallic nanoparticles has been gaining importance since the time people realized the possible toxicity of chemically synthesized nanoparticles [15–18]. “Environmentally benign” biological sources are nowadays successfully employed to produce ecofriendly
AgNPs [19]. Biologically mediated nanoparticle synthesis provides capping or stabilizing agents on the surface of the nanoparticles and prevents the particles from agglomeration. Many phytochemicals of plant origin brings about reduction of metals in ionic form to metallic nanoparticle and can help in overcoming the deleterious consequences of chemically synthesized AgNPs [20–22]. N. arbortristis commonly known as “Parijat, Sewali” or “Harsingar” in India owes great importance in the traditional Indian medicinal system [23]. The flowers are edible, antimicrobial, antimalarial, antispasmodic, antihelminthic, antidepressant and comprise of other important phytochemicals such as anti-oxidants, phenolic compounds and flavonoids [24–30]. Alcoholic extract of N. arbortristis flowers has been reported to possess strong reducing power [31]. Ethanolic flower extract (EFE) of N. arbortristis has already been reported to reduce gold (Au+) ions to metallic gold nanoparticles [32]. 2. Materials and methods 2.1. Materials
⁎ Corresponding author at: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. E-mail addresses: [email protected], [email protected] (U. Bora).
http://dx.doi.org/10.1016/j.msec.2014.10.069 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Mouse fibroblastic cells (L929) were purchased from the National Centre for Cell Science (NCCS), Pune, India. AgNO3 of analytical grade
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was purchased from Merck (Mumbai, India) and ethanol was obtained from Helix India (Guwahati, India). N. arbortristis flowers were collected from a locality near IIT Guwahati and Majuli River Island. Flowers were shade dried at room temperature for about one month. Dried flowers were grinded and sieved to obtain fine powder. One gram of the powder was extracted in 10 mL of ethanol under incubation cum mild shaking condition at room temperature (25 °C). After 72 h, the ethanolic flower extract (EFE) was purified by double filtration (Whatman filter paper). The filtered EFE obtained was directly used for the synthesis of AgNPs as well as stored at 4 °C for further analyses.
2.2. Green synthesis of AgNPs For synthesis of AgNPs, EFE concentration of 5% (v/v) was treated with 1 mM aqueous solution of AgNO 3 with a reaction volume of 500 μl and final volume made up to 2 mL with double distilled water. The reaction mixture was subjected to mild stirring (CMAG-HS7, IKA®) of 200 rpm at 80 °C and observed for color change. Reaction parameters were optimized by varying the volume of EFE (1–10%), (v/v) against 1 mM AgNO 3 , molar concentration kept fixed at 80 °C.
2.3. Characterization of the AgNPs 2.3.1. UV–visible spectroscopy All UV–visible (UV–vis) spectroscopic studies were carried out on Cary 100 BIO UV–vis spectrophotometer (Varian, Palo Alto, CA, USA), to find out the surface plasmonic resonance (SPR) of the AgNPs.
2.3.2. Transmission electron microscope (TEM) Sample preparation for TEM analysis includes centrifugation of synthesized AgNP colloidal solution (5 mL) twice at 20,000 rpm for 20 min to remove the non-covalently bounded molecules on their surfaces. The resulting pellet was redispersed in 1 mL of distilled water, a few drops were placed over a carbon-coated copper grid and the water was evaporated in a hot air oven (Daihan Labtech Co. Ltd. model LDO-150F, New Delhi, India) at 60 °C for 4 h. Transmission electron microscope (TEM) measurements were performed on a TEM instrument (JEOL model 2100, JEOL Ltd., Tokyo, Japan) operated at 190 V of 200 kV.
2.3.3. XRD, TGA, DTA and FT-IR analyses To obtain the X-ray diffraction (XRD) pattern, AgNP solution was placed on a microscope glass slide and allowed to dry in a hot air oven at 50 °C, and the process was repeated to form a layer on the glass slide. The dried samples were analyzed with the help of an XRD instrument (Bruker Advance D8 XRD machine, Bruker, Madison, WI, USA) with a Cu source at 1.5406 Å wavelengths in thin film mode. For FTIR analysis, AgNP colloidal solution (50 mL) was synthesized with optimum parameters (5% of EFE, 1 mM AgNO3) and centrifuged at 20,000 rpm for 20 min. The resulting pellet was resuspended in 5 mL of distilled water and lyophilized (Christ Gefriertrocknungsanlagen GmbH Model 1–4, Osterode, Germany) for 16 h. Infrared spectra were recorded using a Fourier transform infrared (FT-IR) spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) from 4000/cm to 450/cm, with a resolution of 2 cm and five scans/sample by using 1 mg of finely powdered AgNPs prepared with 200 mg of KBr. 5 mg of lyophilized AgNPs was used for thermogravimetric (TGA) and differential thermal analysis (DTA) analyses. 2.4. Antioxidant assay Antioxidant activity of the plant material was determined by DPPH scavenging assay with ascorbic acid as a standard followed by experimental analysis for presence of total phenolic compounds with gallic acid as a standard was performed according to SánchezMoreno et al. and Chang et al. respectively [33,34]. 2.5. Antimicrobial assay In vitro antibacterial activity was evaluated by using agar well diffusion assay with Mueller Hinton Agar (MHA) as growth media and determination of zone of inhibition measurement in millimeters [35]. Antibacterial activity was evaluated against gram negative bacteria Escherichia coli MTCC 443 at different concentrations of AgNPs synthesized from the N. arbortristis EFE. Fresh overnight cultures as inoculums of E. coli MTCC 443 (100 μl) were seeded on MHA plates using sterile cotton swabs. Agar media were bored with a sterile gel borer to create wells of 5 mm in diameter. 100 μl of different concentrations of AgNPs (50, 150, 250 and 500 μg/mL) were poured into separate wells and plates were incubated at 37 °C for 24 h. The diameters of inhibition zones were used to determine the antimicrobial activity and the average of 3 replicas was calculated. Growth curve of AgNP treated E. coli MTCC
Fig. 1. (1a) UV–visible absorption spectra of AgNPs synthesized with different concentrations of EFE (1–10%) against 1 mM AgNO3 at 80 °C. (1b) The SPR peak intensities against different concentrations of EFE (1–10%).
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Fig. 2. (A) TEM, (B) Particle size distribution and (C) UHRTEM of AgNPs synthesized at optimum concentration (5% EFE) at 80 °C.
443 was obtained as absorbance vs concentration at an optical density of 600 nm after inoculating 103 CFU/mL cells in Mueller Hinton broth. The results were further confirmed by morphology study under FESEM (Zeiss, Sigma). 2.6. Cell viability assay Mouse fibroblastic cells (L929) were cultured and maintained according to supplier guidelines. Cytotoxicity of the synthesized AgNPs was assessed by MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5diphenyl tetrazolium bromide) dye conversion assay [20]. L929 cells at a density of 1 × 104 per well were cultured in 100 μl of cell culture medium (DMEM: Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum in a 96-well cell culture plate. After 24 h, cultured cells were treated with a series of concentrations (5, 10, 20, 40, 50, 100 200, and 250 μg/mL) of filter sterilized AgNPs in 100 μl/well (culture medium: DMEM without serum) and incubated further for 24 h. This was followed by removal of the medium and treatment with MTT dye at a final concentration of (0.5 mg/mL) and further incubated for 4 h. Finally, 100 μl of dimethylsulfoxide (DMSO) was added to each well to dissolve blue formazan precipitate, and absorbance was measured at 570 nm using a microplate reader (Bio-Rad Model 680; Bio-Rad). Cell viability was expressed as a percentage of the control by the following equation:
where, Nt and Nc represent the absorbance of AgNP treated and control cells respectively (N = 5; where N is the no. of independent experiments). For enumeration of dead cells, Acridine orange (AO) and Ethidium bromide (EB) staining were applied to both treated and untreated live and dead cells (L929). In this study, 5 μg/mL and 250 μg/mL of AgNPs were taken as minimum and maximum concentrations respectively for the toxicity study. The documentation of cell viability was carried out under an inverted microscope (Eclipse TS100, Nikon) equipped with fluorescence unit and digital camera (Coolpix 5400, Nikon).
3. Results and discussions 3.1. Reducing capability of N. arbortristis EFE The reducing efficiency of N. arbortristis EFE was indicated by successful appearance of brown color which occurs due to the reduction of ionic silver to metallic AgNPs. The phytochemicals occurring in the EFE were responsible for the reduction of Ag+ ions to AgNPs [30,31]. The capping of phytochemicals on the surface of the AgNPs was evident from FTIR studies.
Viabilityð%Þ ¼ Nt =Nc 100
Fig. 3. XRD pattern of AgNPs synthesized using N. arbortristis EFE.
Fig. 4. FTIR spectrum of lyophilized N. arbortristis EFE (spectrum 1) and AgNPs synthesized with N. arbortristis EFE (spectrum 2).
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Fig. 5. TGA and DTA spectra of synthesized AgNPs.
Table 1 Antioxidant activity and IC50 values of EFE and AgNPs. Concentration of EFE (μg/mL)
% Inhibition
IC50 for antioxidant activity of EFE (μg/mL)
Concentration of AgNPs (μg/mL)
% Inhibition
IC50 for antioxidant activity of AgNPs (μg/mL)
5 10 20 40 50 100
17.54 37.06 50.01 58.09 65.66 75.61
20.01 ± 0.29
5 10 20 40 50 100
9.25 11.66 20.56 33.81 38.52 42.46
108.21 ± 3.20
± ± ± ± ± ±
0.40 0.99 0.66 0.86 0.89 1.51
± ± ± ± ± ±
0.73 0.33 0.80 0.14 0.73 0.65
3.2. Physical and optical properties of the AgNPs 3.2.1. UV–visible spectroscopic analysis UV–visible spectra revealed the excitation of surface plasmon vibrations of synthesized AgNPs at a lower wavelength undergoing a blue shift. The SPR peaks at lower concentrations of 1–2% (v/v) of the EFE were broad whereas with slightly increasing concentration, the SPR peaks became sharp and narrower. At a very higher concentration of EFE (8–10%), the SPR band broadened like that of 1–2% (v/v) of EFE concentration indicating that at very higher and lower concentrations the
Table 2 Total phenolic content in EFE. Concentration of EFE (μg/mL)
Total phenolic content (μg/mL)
5 10 20 40 50 100 150 250 500
12.04 21.90 43.20 107.70 203.35 385.96 441.32 470.88 704.51
± ± ± ± ± ± ± ± ±
0.25 0.38 0.81 2.23 0.75 2.66 3.03 2.26 1.61
Fig. 6. Antibacterial assay: inhibition zone for E. coli; (A) negative control, (B) positive control; treatment with different concentrations (50, 150, 250 and 500 μg/mL) of streptomycin, (C) test; treatment with different concentrations (50, 150, 250 and 500 μg/mL) of AgNPs.
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Table 3 Antibacterial activity of AgNPs synthesized from N. arbortristis EFE against E. coli 443. Concentration of streptomycin (μg)
Diameter of zone of inhibition (cm) (mean ± SE)
Concentration AgNPs (μg/mL)
Diameter of zone of inhibition (cm) (mean ± SE)
50 150 250 500
1.10 1.57 1.73 2.07
50 150 250 500
No zone of inhibition No zone of inhibition Partial zone is formed Clear zone with 1.47 ± 0.18
± ± ± ±
0.15 0.03 0.03 0.12
3.2.3. X-ray diffraction (XRD) studies AgNPs on X-Ray diffraction showed four Bragg reflections depicting the fcc structure of the synthesized nanoparticles. The diffraction peaks of the XRD pattern linked to their relative intensities as (111), (200), (220) and (311) for 38.1°, 44.4°, 64.8° and 78° respectively confirming the crystalline structure of the AgNPs (Fig. 3) [37].
Fig. 7. Growth curve of AgNPs treated E. coli cells (Absorbance at 600 nm).
nanoparticle formation is anisotropic (Fig. 1a). The colloidal solutions having anisotropic phenomenon usually gives the broadening of peaks as different sized nanoparticles exhibit different optical properties. The SPR peak intensities explained the logarithmic trend in formation of the AgNPs from lower to higher concentration of EFE (Fig. 1b). 3.2.2. Transmission electron microscope (TEM) analysis AgNPs synthesized using 5% (v/v) EFE which showed the desired SPR peak was selected for the TEM analysis. The TEM of the AgNPs clearly exhibited the morphology of the particles being spherical and oval in shape (Fig. 2A). We applied the UHRTEM to see the AgNPs at the atomic level. The size of synthesized AgNPs ranges from 5–20 nm (Fig. 2B). UHRTEM is a powerful tool to study properties of materials on the atomic scale, such as semiconductors, metals, nanoparticles and sp2-bonded carbon (e.g. graphene, C nanotubes). We observe the lattice fringes on the surface of the AgNPs under ultra high resolution TEM (UHRTEM) which are in accordance with the silver metal [Fig. 2C] [36]. The uniform shapes acquired by the AgNPs explain that there was no secondary nucleation or agglomeration in the synthesis process.
3.2.4. Capping of AgNPs by phytochemicals occurring in N. arbortristis EFE FTIR analysis of the lyophilized EFE displayed a spectrum of distinct IR bands characteristic of hydroxyl (3220–3250 cm−1), alkanes (715, 2895 & 2960 cm−1), C_C of benzene (1627 cm−1), aromatic amines (1374 cm− 1) and aliphatic amines (1040 & 1053 cm− 1) functional groups (Fig. 4, spectra 1) [38]. The alcoholic extract of N. arbortristis flowers were previously reported to contain phytochemicals as phenolics, flavonoids, tannins, terpenoids, saponins and phlobatannins [39]. A similar kind of IR bands for the lyophilized AgNPs were obtained at 3430 cm− 1 (OH), 2917 cm− 1 & 2848 cm− 1 (alkanes), 1696 cm− 1 (carbonyl), 1435 cm−1 (geminal methyl) and 1044 cm−1 (aliphatic amines) (Fig. 4, spectra 2). The appearance of these bands confirms the capping of AgNPs by the phytochemicals present in the EFE. The minor shifts in the band spectrum of Fig. 4 (spectra 2) from the spectra 1 is due to the interaction of EFE with AgNPs which changed the original transmittance level of EFE.
3.3. TGA and DTA analyses We found out the amount (%) of organic material coated on the AgNPs by TGA/DTA analysis. It was observed that 70% of organic material was degraded due to the higher temperature. TGA spectrum of AgNPs occurs over a wide temperature range (195–515 °C) which revealed the significant weight loss (70%) of AgNPs (Fig. 5). This clearly indicated that bioactive molecules were capped on the AgNPs and were degraded due to high temperature. Hence, it was deduced that AgNPs were capped with bioactive molecules originating from EFE.
Fig. 8. FESEM micrograph showing morphological difference between AgNP treated and untreated E. coli cells. (7a) Untreated E. coli cells. (7b) Treated E.coli cells showing maximum damage and rupture of cell walls.
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Fig. 9. Morphology of untreated and treated L929 cells; (9a) control, (9b) L929 cells treated with 5 μg/ml AgNPs, (9c) L929 cells treated with 250 μg/mL AgNPs.
3.4. Antioxidant activity evaluation of EFE and AgNPs The antioxidant activity of the EFE was measured on the basis of the scavenging activity of the stable 1,1-diphenyl 2-picrylhydrazyl (DPPH) free radical. DPPH represents the standard and position of electron paramagnetic resonance signals and among the major applications of DDPH is monitoring of free radicals in antioxidant assays. Antioxidant activity was measured as disappearance of color at an absorbance of 517 nm (Table 1). Ascorbic acid was used as standard reference for estimation of antioxidant activity. IC50 value was calculated from a regression plot bearing %
disappearance of color vs. concentration of sample (50–500 μg/mL). IC50 is designated as the amount of sample required to decrease 50% of the initial DPPH concentration added (Lim, 2007). The IC50 values for EFE and AgNPs are summarized in Table 1. This clearly depicts the presence of reducing phytochemicals in the EFE which reduced the silver ions into the corresponding nanoparticles. In addition to it, FTIR data of the lyophilized AgNPs strongly supports the occurrence of phytochemicals in the EFE and responsible for the synthesis of the AgNPs (Fig. 4, spectra 2). We have evaluated the presence of total phenolic compounds at different concentrations of the EFE using gallic acid as a standard, the results are summarized in Table 2. Total phenolic content in EFE concentration of 5–500 µg/mL was found to lie between 12.04 ± 0.25 µg/mL and 704.51 ± 1.61 µg/mL. 3.5. Antibacterial activity of synthesized AgNPs
Fig. 10. Cell viability assay: L929 cells showed 83% viability at the 250 μg/mL of AgNPs.
E. coli MTCC 443 has been utilized as a model organism by many researchers for antibacterial studies using green synthesized AgNPs [40]. Based on the results from the agar well diffusion assay, AgNPs with concentration of 500 μg/mL presented a significant inhibition activity against E. coli MTCC 443. The clear inhibition zone surrounding the bore having a radius of 1.47 ± 0.18 cm illustrates the antimicrobial activity of the AgNPs (Fig. 6C). However, we did not observed any clear zone formation in negative control. The minimum inhibitory concentration (MIC) of the AgNPs was evaluated as the lowest concentration of AgNP that inhibited the growth of pathogenic E. coli MTCC 443. Agar well diffusion method (MHA) involving streptomycin as a positive control exhibited clear zones of inhibition in the range of 50–500 μg/mL (Table 3). The MIC calculated for the inhibition of bacteria with reference to streptomycin is 220.05 μg/mL of AgNPs.
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Initially, AgNPs capped with polyphenolic compounds damage the cell membranes of bacteria, most effectively on Gram-negative bacteria by producing reactive oxygen species and free radicals that induce membrane damage [41]. Once the membranes are damaged, the membrane potential is disturbed by loss of intracellular K+ ions. Further consequences lead to cytoplasmic leakage and finally the release of lipopolysaccharide molecules and membrane proteins. The lipid bilayer of the outer membrane is asymmetric and composed of lipopolysaccharide whereas the inner wall is close-packed phospholipid chains exhibiting turbulence in membranous permeability. According to Kim et al (2007), AgNPs being smaller in size accumulate on the bacterial membranes to form irregular pits that initiate the leakage of cell components. Apart from these physiological mechanisms, the capping of AgNPs with EFE may supplement additional antimicrobial activity [41]. The antimicrobial activity was correspondingly confirmed by growth curve study of E. coli MTCC 443 treated with several concentrations of the AgNPs. 500 μg/mL of AgNP treated bacterial cells (103 CFU/mL) showed maximum growth inhibition in a time period of 24 h incubated in MH broth at 37 °C (Fig. 7). Field Emission Scanning Electron Microscopy (FESEM) imaging also proved the damage of bacterial cells after being treated with variable concentrations of AgNPs (Fig. 8b). 3.6. Cytotoxicity and cell viability studies In MTT dye conversion cytotoxicity assay, the activity of mitochondrial dehydrogenase of live mouse fibroblast cells (L929) cleaved the tetrazolium ring in the tetrazolium salts. Only active mitochondria contain these enzymes and therefore the reaction occurs only in living cells. Treated L929 cells with different concentrations of AgNPs for 24 h showed that the AgNPs did not cause significant loss in cell viability compared to the control or untreated cells (Fig. 9a, b, c). The AgNP treated L929 cells showed almost 83% viability at highest concentration (250 μg/mL) after 24 h of treatment. This confirmed that the synthesized AgNPs were biocompatible and can be used for biomedical applications (Fig. 10). Viability stains proved the membrane integrity of L929 cells based on the uptake or exclusion of a dye from the cells. Acridine Orange (AO) is a membrane-permeable dye that stains both live and dead cells, while Ethidium bromide (EB) passes through membrane of dead and dying cells in untreated as well as treated cells. AO stained cells emitted green color and EB staining turned the cells red. The untreated L929 cells gave intense green fluorescence and weak signals for red fluorescence which was expected for the control experiment (Fig. 9a). Cells treated with the lowest (5 μg/mL) and highest (250 μg/mL) concentrations of AgNPs retained their viability confirmed by AO permeability causing a strong green fluorescence but a weak fluorescence for EB (Fig. 9b, c). 4. Conclusion We investigated the reducing capabilities of N. arbortristis EFE for the synthesis of AgNPs. Phytochemicals such as antioxidants and phenolic compounds present in N. arbortristis are involved in the reduction of silver ions to AgNPs. TEM monograph indicated the smaller size of AgNPs desirable for most of the therapeutic applications and water treatment processes. The capping of AgNPs by phytochemicals present in the flower extract was evident from FTIR studies. The crystalline nature of the AgNPs was confirmed from the SAED and XRD pattern. The efficacy of AgNPs was proved by in vitro antibacterial, cytotoxicity and cell viability studies. The antibacterial activity of AgNPs was found to be efficient against pathogenic gram negative bacteria (E. coli MTCC 443). The cytotoxicity studies revealed that the maximum dose (250 μg/mL) of
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synthesized AgNPs showed insignificant toxicity on L929 cells. This eco-friendly method for the synthesis of AgNPs provides an opportunity to use these AgNPs for application in drug delivery and molecular imaging.
Acknowledgments Authors are thankful to DBT, Government of India for funding the project, Institutional Biotech Hub, Centre for the Environment, IIT Guwahati (Project No: BT/04/NE/2009) under which the work was carried out.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Green synthesis and characterization of silver nanoparticles using alcoholic flower extract of Nyctanthes arbortristis and in vitro investigation of their antibacterial and cytotoxic activities Nayanmoni Gogoi a,c, Punuri Jayasekhar Babu a,b, Chandan Mahanta a,d, Utpal Bora a,b,⁎ a
Biotech Hub, Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India Biomaterials and Tissue Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India c Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India d Department of Civil Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India b
a r t i c l e
i n f o
Article history: Received 30 June 2014 Received in revised form 19 September 2014 Accepted 23 October 2014 Available online 24 October 2014 Keywords: Nyctanthes arbortristis Spectroscopy Crystal growth of silver nanoparticles FTIR Electron microscopy Nanocrystalline materials Antibacterial Cell viability
a b s t r a c t Here we report the synthesis of silver nanoparticles using ethanolic flower extract of Nyctanthes arbortristis, UVvisible spectra and TEM indicated the successful formation of silver nanoparticles. Crystalline nature of the silver nanoparticles was confirmed by X-ray diffraction. Fourier Transform Infra-Red Spectroscopy analysis established the capping of the synthesized silver nanoparticles with phytochemicals naturally occurring in the ethanolic flower extract of N. arbortristis. The synthesized silver nanoparticles showed antibacterial activity against the pathogenic strain of Escherichia coli MTCC 443. Furthermore, cytotoxicity of the silver nanoparticles was tested on mouse fibroblastic cell line (L929) and found to be non-toxic, which thus proved their biocompatibility. Antibacterial activity and cytotoxicity assay carried out in this study open up an important perspective of the synthesized silver nanoparticles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silver nanoparticles (AgNPs) have been the focus of increasing research interest in the past decade. In the recent times, AgNPs serve as an excellent candidate for most of the therapeutic purposes [1]. Studies on AgNPs have confirmed their potential application in many fields of science [2]. AgNPs have shown positive results in wound healing, retinal therapies, DNA sequencing and pharmaceuticals along with other conventional uses like in electronics, optics, catalysis and Raman scattering [3–11]. The fundamental uses of AgNPs have been highly recognized in water treatment process. Contemporary AgNP filters are effective and utilized against contamination in water treatment and filtration processes [12,13]. The efficiency of such filters is strongly attributed to the antimicrobial properties acquired by AgNPs [14]. Green synthesis of metallic nanoparticles has been gaining importance since the time people realized the possible toxicity of chemically synthesized nanoparticles [15–18]. “Environmentally benign” biological sources are nowadays successfully employed to produce ecofriendly
AgNPs [19]. Biologically mediated nanoparticle synthesis provides capping or stabilizing agents on the surface of the nanoparticles and prevents the particles from agglomeration. Many phytochemicals of plant origin brings about reduction of metals in ionic form to metallic nanoparticle and can help in overcoming the deleterious consequences of chemically synthesized AgNPs [20–22]. N. arbortristis commonly known as “Parijat, Sewali” or “Harsingar” in India owes great importance in the traditional Indian medicinal system [23]. The flowers are edible, antimicrobial, antimalarial, antispasmodic, antihelminthic, antidepressant and comprise of other important phytochemicals such as anti-oxidants, phenolic compounds and flavonoids [24–30]. Alcoholic extract of N. arbortristis flowers has been reported to possess strong reducing power [31]. Ethanolic flower extract (EFE) of N. arbortristis has already been reported to reduce gold (Au+) ions to metallic gold nanoparticles [32]. 2. Materials and methods 2.1. Materials
⁎ Corresponding author at: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. E-mail addresses: [email protected], [email protected] (U. Bora).
http://dx.doi.org/10.1016/j.msec.2014.10.069 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Mouse fibroblastic cells (L929) were purchased from the National Centre for Cell Science (NCCS), Pune, India. AgNO3 of analytical grade
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was purchased from Merck (Mumbai, India) and ethanol was obtained from Helix India (Guwahati, India). N. arbortristis flowers were collected from a locality near IIT Guwahati and Majuli River Island. Flowers were shade dried at room temperature for about one month. Dried flowers were grinded and sieved to obtain fine powder. One gram of the powder was extracted in 10 mL of ethanol under incubation cum mild shaking condition at room temperature (25 °C). After 72 h, the ethanolic flower extract (EFE) was purified by double filtration (Whatman filter paper). The filtered EFE obtained was directly used for the synthesis of AgNPs as well as stored at 4 °C for further analyses.
2.2. Green synthesis of AgNPs For synthesis of AgNPs, EFE concentration of 5% (v/v) was treated with 1 mM aqueous solution of AgNO 3 with a reaction volume of 500 μl and final volume made up to 2 mL with double distilled water. The reaction mixture was subjected to mild stirring (CMAG-HS7, IKA®) of 200 rpm at 80 °C and observed for color change. Reaction parameters were optimized by varying the volume of EFE (1–10%), (v/v) against 1 mM AgNO 3 , molar concentration kept fixed at 80 °C.
2.3. Characterization of the AgNPs 2.3.1. UV–visible spectroscopy All UV–visible (UV–vis) spectroscopic studies were carried out on Cary 100 BIO UV–vis spectrophotometer (Varian, Palo Alto, CA, USA), to find out the surface plasmonic resonance (SPR) of the AgNPs.
2.3.2. Transmission electron microscope (TEM) Sample preparation for TEM analysis includes centrifugation of synthesized AgNP colloidal solution (5 mL) twice at 20,000 rpm for 20 min to remove the non-covalently bounded molecules on their surfaces. The resulting pellet was redispersed in 1 mL of distilled water, a few drops were placed over a carbon-coated copper grid and the water was evaporated in a hot air oven (Daihan Labtech Co. Ltd. model LDO-150F, New Delhi, India) at 60 °C for 4 h. Transmission electron microscope (TEM) measurements were performed on a TEM instrument (JEOL model 2100, JEOL Ltd., Tokyo, Japan) operated at 190 V of 200 kV.
2.3.3. XRD, TGA, DTA and FT-IR analyses To obtain the X-ray diffraction (XRD) pattern, AgNP solution was placed on a microscope glass slide and allowed to dry in a hot air oven at 50 °C, and the process was repeated to form a layer on the glass slide. The dried samples were analyzed with the help of an XRD instrument (Bruker Advance D8 XRD machine, Bruker, Madison, WI, USA) with a Cu source at 1.5406 Å wavelengths in thin film mode. For FTIR analysis, AgNP colloidal solution (50 mL) was synthesized with optimum parameters (5% of EFE, 1 mM AgNO3) and centrifuged at 20,000 rpm for 20 min. The resulting pellet was resuspended in 5 mL of distilled water and lyophilized (Christ Gefriertrocknungsanlagen GmbH Model 1–4, Osterode, Germany) for 16 h. Infrared spectra were recorded using a Fourier transform infrared (FT-IR) spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) from 4000/cm to 450/cm, with a resolution of 2 cm and five scans/sample by using 1 mg of finely powdered AgNPs prepared with 200 mg of KBr. 5 mg of lyophilized AgNPs was used for thermogravimetric (TGA) and differential thermal analysis (DTA) analyses. 2.4. Antioxidant assay Antioxidant activity of the plant material was determined by DPPH scavenging assay with ascorbic acid as a standard followed by experimental analysis for presence of total phenolic compounds with gallic acid as a standard was performed according to SánchezMoreno et al. and Chang et al. respectively [33,34]. 2.5. Antimicrobial assay In vitro antibacterial activity was evaluated by using agar well diffusion assay with Mueller Hinton Agar (MHA) as growth media and determination of zone of inhibition measurement in millimeters [35]. Antibacterial activity was evaluated against gram negative bacteria Escherichia coli MTCC 443 at different concentrations of AgNPs synthesized from the N. arbortristis EFE. Fresh overnight cultures as inoculums of E. coli MTCC 443 (100 μl) were seeded on MHA plates using sterile cotton swabs. Agar media were bored with a sterile gel borer to create wells of 5 mm in diameter. 100 μl of different concentrations of AgNPs (50, 150, 250 and 500 μg/mL) were poured into separate wells and plates were incubated at 37 °C for 24 h. The diameters of inhibition zones were used to determine the antimicrobial activity and the average of 3 replicas was calculated. Growth curve of AgNP treated E. coli MTCC
Fig. 1. (1a) UV–visible absorption spectra of AgNPs synthesized with different concentrations of EFE (1–10%) against 1 mM AgNO3 at 80 °C. (1b) The SPR peak intensities against different concentrations of EFE (1–10%).
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Fig. 2. (A) TEM, (B) Particle size distribution and (C) UHRTEM of AgNPs synthesized at optimum concentration (5% EFE) at 80 °C.
443 was obtained as absorbance vs concentration at an optical density of 600 nm after inoculating 103 CFU/mL cells in Mueller Hinton broth. The results were further confirmed by morphology study under FESEM (Zeiss, Sigma). 2.6. Cell viability assay Mouse fibroblastic cells (L929) were cultured and maintained according to supplier guidelines. Cytotoxicity of the synthesized AgNPs was assessed by MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5diphenyl tetrazolium bromide) dye conversion assay [20]. L929 cells at a density of 1 × 104 per well were cultured in 100 μl of cell culture medium (DMEM: Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum in a 96-well cell culture plate. After 24 h, cultured cells were treated with a series of concentrations (5, 10, 20, 40, 50, 100 200, and 250 μg/mL) of filter sterilized AgNPs in 100 μl/well (culture medium: DMEM without serum) and incubated further for 24 h. This was followed by removal of the medium and treatment with MTT dye at a final concentration of (0.5 mg/mL) and further incubated for 4 h. Finally, 100 μl of dimethylsulfoxide (DMSO) was added to each well to dissolve blue formazan precipitate, and absorbance was measured at 570 nm using a microplate reader (Bio-Rad Model 680; Bio-Rad). Cell viability was expressed as a percentage of the control by the following equation:
where, Nt and Nc represent the absorbance of AgNP treated and control cells respectively (N = 5; where N is the no. of independent experiments). For enumeration of dead cells, Acridine orange (AO) and Ethidium bromide (EB) staining were applied to both treated and untreated live and dead cells (L929). In this study, 5 μg/mL and 250 μg/mL of AgNPs were taken as minimum and maximum concentrations respectively for the toxicity study. The documentation of cell viability was carried out under an inverted microscope (Eclipse TS100, Nikon) equipped with fluorescence unit and digital camera (Coolpix 5400, Nikon).
3. Results and discussions 3.1. Reducing capability of N. arbortristis EFE The reducing efficiency of N. arbortristis EFE was indicated by successful appearance of brown color which occurs due to the reduction of ionic silver to metallic AgNPs. The phytochemicals occurring in the EFE were responsible for the reduction of Ag+ ions to AgNPs [30,31]. The capping of phytochemicals on the surface of the AgNPs was evident from FTIR studies.
Viabilityð%Þ ¼ Nt =Nc 100
Fig. 3. XRD pattern of AgNPs synthesized using N. arbortristis EFE.
Fig. 4. FTIR spectrum of lyophilized N. arbortristis EFE (spectrum 1) and AgNPs synthesized with N. arbortristis EFE (spectrum 2).
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Fig. 5. TGA and DTA spectra of synthesized AgNPs.
Table 1 Antioxidant activity and IC50 values of EFE and AgNPs. Concentration of EFE (μg/mL)
% Inhibition
IC50 for antioxidant activity of EFE (μg/mL)
Concentration of AgNPs (μg/mL)
% Inhibition
IC50 for antioxidant activity of AgNPs (μg/mL)
5 10 20 40 50 100
17.54 37.06 50.01 58.09 65.66 75.61
20.01 ± 0.29
5 10 20 40 50 100
9.25 11.66 20.56 33.81 38.52 42.46
108.21 ± 3.20
± ± ± ± ± ±
0.40 0.99 0.66 0.86 0.89 1.51
± ± ± ± ± ±
0.73 0.33 0.80 0.14 0.73 0.65
3.2. Physical and optical properties of the AgNPs 3.2.1. UV–visible spectroscopic analysis UV–visible spectra revealed the excitation of surface plasmon vibrations of synthesized AgNPs at a lower wavelength undergoing a blue shift. The SPR peaks at lower concentrations of 1–2% (v/v) of the EFE were broad whereas with slightly increasing concentration, the SPR peaks became sharp and narrower. At a very higher concentration of EFE (8–10%), the SPR band broadened like that of 1–2% (v/v) of EFE concentration indicating that at very higher and lower concentrations the
Table 2 Total phenolic content in EFE. Concentration of EFE (μg/mL)
Total phenolic content (μg/mL)
5 10 20 40 50 100 150 250 500
12.04 21.90 43.20 107.70 203.35 385.96 441.32 470.88 704.51
± ± ± ± ± ± ± ± ±
0.25 0.38 0.81 2.23 0.75 2.66 3.03 2.26 1.61
Fig. 6. Antibacterial assay: inhibition zone for E. coli; (A) negative control, (B) positive control; treatment with different concentrations (50, 150, 250 and 500 μg/mL) of streptomycin, (C) test; treatment with different concentrations (50, 150, 250 and 500 μg/mL) of AgNPs.
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Table 3 Antibacterial activity of AgNPs synthesized from N. arbortristis EFE against E. coli 443. Concentration of streptomycin (μg)
Diameter of zone of inhibition (cm) (mean ± SE)
Concentration AgNPs (μg/mL)
Diameter of zone of inhibition (cm) (mean ± SE)
50 150 250 500
1.10 1.57 1.73 2.07
50 150 250 500
No zone of inhibition No zone of inhibition Partial zone is formed Clear zone with 1.47 ± 0.18
± ± ± ±
0.15 0.03 0.03 0.12
3.2.3. X-ray diffraction (XRD) studies AgNPs on X-Ray diffraction showed four Bragg reflections depicting the fcc structure of the synthesized nanoparticles. The diffraction peaks of the XRD pattern linked to their relative intensities as (111), (200), (220) and (311) for 38.1°, 44.4°, 64.8° and 78° respectively confirming the crystalline structure of the AgNPs (Fig. 3) [37].
Fig. 7. Growth curve of AgNPs treated E. coli cells (Absorbance at 600 nm).
nanoparticle formation is anisotropic (Fig. 1a). The colloidal solutions having anisotropic phenomenon usually gives the broadening of peaks as different sized nanoparticles exhibit different optical properties. The SPR peak intensities explained the logarithmic trend in formation of the AgNPs from lower to higher concentration of EFE (Fig. 1b). 3.2.2. Transmission electron microscope (TEM) analysis AgNPs synthesized using 5% (v/v) EFE which showed the desired SPR peak was selected for the TEM analysis. The TEM of the AgNPs clearly exhibited the morphology of the particles being spherical and oval in shape (Fig. 2A). We applied the UHRTEM to see the AgNPs at the atomic level. The size of synthesized AgNPs ranges from 5–20 nm (Fig. 2B). UHRTEM is a powerful tool to study properties of materials on the atomic scale, such as semiconductors, metals, nanoparticles and sp2-bonded carbon (e.g. graphene, C nanotubes). We observe the lattice fringes on the surface of the AgNPs under ultra high resolution TEM (UHRTEM) which are in accordance with the silver metal [Fig. 2C] [36]. The uniform shapes acquired by the AgNPs explain that there was no secondary nucleation or agglomeration in the synthesis process.
3.2.4. Capping of AgNPs by phytochemicals occurring in N. arbortristis EFE FTIR analysis of the lyophilized EFE displayed a spectrum of distinct IR bands characteristic of hydroxyl (3220–3250 cm−1), alkanes (715, 2895 & 2960 cm−1), C_C of benzene (1627 cm−1), aromatic amines (1374 cm− 1) and aliphatic amines (1040 & 1053 cm− 1) functional groups (Fig. 4, spectra 1) [38]. The alcoholic extract of N. arbortristis flowers were previously reported to contain phytochemicals as phenolics, flavonoids, tannins, terpenoids, saponins and phlobatannins [39]. A similar kind of IR bands for the lyophilized AgNPs were obtained at 3430 cm− 1 (OH), 2917 cm− 1 & 2848 cm− 1 (alkanes), 1696 cm− 1 (carbonyl), 1435 cm−1 (geminal methyl) and 1044 cm−1 (aliphatic amines) (Fig. 4, spectra 2). The appearance of these bands confirms the capping of AgNPs by the phytochemicals present in the EFE. The minor shifts in the band spectrum of Fig. 4 (spectra 2) from the spectra 1 is due to the interaction of EFE with AgNPs which changed the original transmittance level of EFE.
3.3. TGA and DTA analyses We found out the amount (%) of organic material coated on the AgNPs by TGA/DTA analysis. It was observed that 70% of organic material was degraded due to the higher temperature. TGA spectrum of AgNPs occurs over a wide temperature range (195–515 °C) which revealed the significant weight loss (70%) of AgNPs (Fig. 5). This clearly indicated that bioactive molecules were capped on the AgNPs and were degraded due to high temperature. Hence, it was deduced that AgNPs were capped with bioactive molecules originating from EFE.
Fig. 8. FESEM micrograph showing morphological difference between AgNP treated and untreated E. coli cells. (7a) Untreated E. coli cells. (7b) Treated E.coli cells showing maximum damage and rupture of cell walls.
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Fig. 9. Morphology of untreated and treated L929 cells; (9a) control, (9b) L929 cells treated with 5 μg/ml AgNPs, (9c) L929 cells treated with 250 μg/mL AgNPs.
3.4. Antioxidant activity evaluation of EFE and AgNPs The antioxidant activity of the EFE was measured on the basis of the scavenging activity of the stable 1,1-diphenyl 2-picrylhydrazyl (DPPH) free radical. DPPH represents the standard and position of electron paramagnetic resonance signals and among the major applications of DDPH is monitoring of free radicals in antioxidant assays. Antioxidant activity was measured as disappearance of color at an absorbance of 517 nm (Table 1). Ascorbic acid was used as standard reference for estimation of antioxidant activity. IC50 value was calculated from a regression plot bearing %
disappearance of color vs. concentration of sample (50–500 μg/mL). IC50 is designated as the amount of sample required to decrease 50% of the initial DPPH concentration added (Lim, 2007). The IC50 values for EFE and AgNPs are summarized in Table 1. This clearly depicts the presence of reducing phytochemicals in the EFE which reduced the silver ions into the corresponding nanoparticles. In addition to it, FTIR data of the lyophilized AgNPs strongly supports the occurrence of phytochemicals in the EFE and responsible for the synthesis of the AgNPs (Fig. 4, spectra 2). We have evaluated the presence of total phenolic compounds at different concentrations of the EFE using gallic acid as a standard, the results are summarized in Table 2. Total phenolic content in EFE concentration of 5–500 µg/mL was found to lie between 12.04 ± 0.25 µg/mL and 704.51 ± 1.61 µg/mL. 3.5. Antibacterial activity of synthesized AgNPs
Fig. 10. Cell viability assay: L929 cells showed 83% viability at the 250 μg/mL of AgNPs.
E. coli MTCC 443 has been utilized as a model organism by many researchers for antibacterial studies using green synthesized AgNPs [40]. Based on the results from the agar well diffusion assay, AgNPs with concentration of 500 μg/mL presented a significant inhibition activity against E. coli MTCC 443. The clear inhibition zone surrounding the bore having a radius of 1.47 ± 0.18 cm illustrates the antimicrobial activity of the AgNPs (Fig. 6C). However, we did not observed any clear zone formation in negative control. The minimum inhibitory concentration (MIC) of the AgNPs was evaluated as the lowest concentration of AgNP that inhibited the growth of pathogenic E. coli MTCC 443. Agar well diffusion method (MHA) involving streptomycin as a positive control exhibited clear zones of inhibition in the range of 50–500 μg/mL (Table 3). The MIC calculated for the inhibition of bacteria with reference to streptomycin is 220.05 μg/mL of AgNPs.
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Initially, AgNPs capped with polyphenolic compounds damage the cell membranes of bacteria, most effectively on Gram-negative bacteria by producing reactive oxygen species and free radicals that induce membrane damage [41]. Once the membranes are damaged, the membrane potential is disturbed by loss of intracellular K+ ions. Further consequences lead to cytoplasmic leakage and finally the release of lipopolysaccharide molecules and membrane proteins. The lipid bilayer of the outer membrane is asymmetric and composed of lipopolysaccharide whereas the inner wall is close-packed phospholipid chains exhibiting turbulence in membranous permeability. According to Kim et al (2007), AgNPs being smaller in size accumulate on the bacterial membranes to form irregular pits that initiate the leakage of cell components. Apart from these physiological mechanisms, the capping of AgNPs with EFE may supplement additional antimicrobial activity [41]. The antimicrobial activity was correspondingly confirmed by growth curve study of E. coli MTCC 443 treated with several concentrations of the AgNPs. 500 μg/mL of AgNP treated bacterial cells (103 CFU/mL) showed maximum growth inhibition in a time period of 24 h incubated in MH broth at 37 °C (Fig. 7). Field Emission Scanning Electron Microscopy (FESEM) imaging also proved the damage of bacterial cells after being treated with variable concentrations of AgNPs (Fig. 8b). 3.6. Cytotoxicity and cell viability studies In MTT dye conversion cytotoxicity assay, the activity of mitochondrial dehydrogenase of live mouse fibroblast cells (L929) cleaved the tetrazolium ring in the tetrazolium salts. Only active mitochondria contain these enzymes and therefore the reaction occurs only in living cells. Treated L929 cells with different concentrations of AgNPs for 24 h showed that the AgNPs did not cause significant loss in cell viability compared to the control or untreated cells (Fig. 9a, b, c). The AgNP treated L929 cells showed almost 83% viability at highest concentration (250 μg/mL) after 24 h of treatment. This confirmed that the synthesized AgNPs were biocompatible and can be used for biomedical applications (Fig. 10). Viability stains proved the membrane integrity of L929 cells based on the uptake or exclusion of a dye from the cells. Acridine Orange (AO) is a membrane-permeable dye that stains both live and dead cells, while Ethidium bromide (EB) passes through membrane of dead and dying cells in untreated as well as treated cells. AO stained cells emitted green color and EB staining turned the cells red. The untreated L929 cells gave intense green fluorescence and weak signals for red fluorescence which was expected for the control experiment (Fig. 9a). Cells treated with the lowest (5 μg/mL) and highest (250 μg/mL) concentrations of AgNPs retained their viability confirmed by AO permeability causing a strong green fluorescence but a weak fluorescence for EB (Fig. 9b, c). 4. Conclusion We investigated the reducing capabilities of N. arbortristis EFE for the synthesis of AgNPs. Phytochemicals such as antioxidants and phenolic compounds present in N. arbortristis are involved in the reduction of silver ions to AgNPs. TEM monograph indicated the smaller size of AgNPs desirable for most of the therapeutic applications and water treatment processes. The capping of AgNPs by phytochemicals present in the flower extract was evident from FTIR studies. The crystalline nature of the AgNPs was confirmed from the SAED and XRD pattern. The efficacy of AgNPs was proved by in vitro antibacterial, cytotoxicity and cell viability studies. The antibacterial activity of AgNPs was found to be efficient against pathogenic gram negative bacteria (E. coli MTCC 443). The cytotoxicity studies revealed that the maximum dose (250 μg/mL) of
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synthesized AgNPs showed insignificant toxicity on L929 cells. This eco-friendly method for the synthesis of AgNPs provides an opportunity to use these AgNPs for application in drug delivery and molecular imaging.
Acknowledgments Authors are thankful to DBT, Government of India for funding the project, Institutional Biotech Hub, Centre for the Environment, IIT Guwahati (Project No: BT/04/NE/2009) under which the work was carried out.
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